-
Multiplex Immunohistochemistry: The Importance of Staining Order
WhenProducing a Validated Protocol
Jihad Syed1, Jack Ashton2, Jesuchristopher Joseph2, Gemma N
Jones2, Christian Slater1, Alan Sharpe2,Garry Ashton1, William
Howat2, Richard Byers1, Helen K Angell2*
1Institute of Cancer Sciences, University of Manchester,
Manchester, UK;2Translational Medicine, Oncology R&D,
AstraZeneca,Cambridge, UK
ABSTRACT
personalised therapies. Integrated digital histological analysis
of tumours provides a better understanding of the
immune microenvironment and the prognostic relationship
associated with the enumeration and distribution of
specific tumour infiltrating lymphocyte (TILs) subpopulations.
To this effect multiplex cell labelling , alongside
multi-spectral imaging (MSI) is an approach increasingly used to
achieve more accurate in-situ TIL phenotyping
and quantification. However, these approaches require full
validation prior to utilisation , which is the
fundamental aim of this study.
multiplex immunofluorescence (IF) protocol for simultaneous MSI
interrogation of up to six immune cell
antigens of interest; CD3, CD8, FOXP3, CD20, PD-L1 and PD1.
Concordance between single- plex chromogenic
immunohistochemistry (IHC) and single- plex IF staining was
first achieved. Subsequently,
compounding factor influencing multiplex-assay validation was
the non-linear and non-uniform effect of extended
times of heat-induced epitope retrieval (HIER), as antibodies
advance in order in a multiplex protocol.
plex staining, the
effect of order of antibody staining, and offers a framework for
the generation of optimised multiplex
immunofluorescent protocols.
Keywords: Multiplex immunofluorescence; Digital histology;
Validation; Immune infiltrates
INTRODUCTION
The Tumour Microenvironment (TME) is important in
tumourprogression and treatment response, leading to development
ofnew targeted therapies [1,2]. Tumour infiltrating
lymphocytes(TILs) are a common feature of solid cancers with their
type,number and spatial distribution all shown to affect
prognosis[3,4]. Similarly, clinically validated quantification of
CD3+lymphocytes and CD8+ cytotoxic T cells has shown
statistical
superiority to the current TNM classification for prediction
ofoverall survival (OS) and disease-free survival (DFS) in
colorectalcancer [1,5,6]. However, to gain a deeper understanding
of theTME, further characterisation is needed,
includingidentification of phenotypically distinct immune
cellpopulations such as dendritic cells, macrophages, natural
killercells, and their state of activation or exhaustion. The
ability tosimultaneously assess multiple cells in situ is dependent
on
Correspondence to: Helen K Angell, Oncology R&D,
AstraZeneca, Darwin Building, Unit 310, Cambridge Science Park,
Milton Road,Cambridge, CB4 0WG, UK, E-mail:
[email protected]
Received: December 14, 2019; Accepted: December 24, 2019;
Published: December 31, 2019
Citation: Syed J, Ashton J, Joseph J, Jones GN, Slater C, Sharpe
A, et al. (2019) Multiplex Immunohistochemistry: The Importance of
StainingOrder When Producing a Validated Protocol. Immunotherapy
(Los Angel) 5: 157. Doi: 10.35248/2471-9552.19.5.157
Copyright: © 2019 Syed J, et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution
License, whichpermits unrestricted use, distribution, and
reproduction in any medium, provided the original author and source
are credited.
1
Aim: The complexity of multifactorial diseases, such as cancer,
poses significant challenges to the development of
Methods: Whole sections and tissue microarrays of
lymphocyte-rich tissue were used to develop and validate a
Results: In methods where multiplexing is enabled using antigen
retrieval to strip prior antibodies, the principal
Conclusion: This study demonstrates the fidelity of multiplex
staining as representative of single-
Immunotherapy: Open AccessResearch Article
Immunotherapy (Los Angel), Vol.5 Iss.2 No:1000157
the effect of the
position in a multiplex IF order for any given antibody was
investigated, understanding the impact of antibody steric
hindrance and antibody stripping conditions.
-
development of accurate, sensitive and quantifiable
multiplexingassays.
Multiplex immunostaining, whether chromogenic orfluorescent, is
becoming increasingly popular with theemergence of robust multiplex
staining methods and advancedmultispectral imaging [7-9]. Multiplex
staining has significantadvantages in terms of preservation of
precious tissue samples,and co-localisation of antigens [10]. The
latter is of particularimportance for the growing field of
immunotherapy, enablingdetection and identification of tissue
infiltrating cells, alongwith characterisation of response
biomarkers such as PD-L1[8,10-13]. However, stringent validation
steps must beimplemented when developing multiplex assays to ensure
theseassays generate comparable data to the gold-standard
single-plexassays.
This project describes development and validation of amultiplex
staining protocol against CD3, CD8, FOXP3, CD20,PD-L1 and PD1,
using a multiplex methodology that includessequential rounds of
antibody stripping. Critically it highlightsthe challenges of
developing multiplex assays and stresses theimportance of not using
a ‘plug and play’ approach, which canresult in inaccurate data
read-outs. Measurement of the fidelityof multiplex staining as
representative of single-plex staining, theeffect of antigen
retrieval time and the order of antibodystaining are critical to
the generation of a robust optimisedmultiplex immunofluorescence
(IF) protocol.
MATERIALS AND METHODS
Tissue cohorts and ethics
Cohort 1: Whole tissue sections of tonsil. Ethical approval
wasprovided by the Central Manchester Multicentre ResearchEthical
Committee (03/TG/076).
Cohort 2: A follicular-lymphoma tissue microarray
(TMA)constructed from 40 archived pre-diagnostic FFPE biopsies
(82cores). Ethical permission for this study was obtained from
theCentral Manchester Multi-Centre Research Ethical
Committee(03/8/106).
Control tissue: Samples were acquired via the AstraZenecaBiobank
[Human Tissue Authority (Licence No. 12109) andNational Research
Ethics Service Committee (NREC) Approvalas a Research Tissue Bank
(RTB) (REC No 17/NW/0207)].
Chromogenic single-plex immunohistochemistrystaining
Chromogenic staining (DAB single-plex) was performed on
theVentana Discovery Ultra autostainer (Roche) using UltraViewDAB
(Roche) or Chromomap DAB (Roche) detection kits. 4 µmwhole tissue
FFPE sections were mounted on SuperFrost™Plusslides and dried
over-night at 37°C prior to staining. TheVentana staining protocol
involved EZ prep deparaffinisation,followed by CC1 antigen
retrieval (pH 9, 95°C), thenendogenous peroxidase blocking (Roche),
followed byincubation with the primary antibodies under the
conditions
was performed using Hematoxylin II (Roche) and then
bluingreagent (Roche).
Staining of single-plex, duplex and
multipleximmunofluorescence
IF staining was performed on the Ventana Discovery
Ultraautostainer, with the 6-plex achieved using three sequential
dual-plex protocols. Tissues were sectioned, deparaffinised,
antigenretrieved and blocked for endogenous peroxidase as
describedabove. The primary antibodies used, and their
incubationdetails, are listed in Table 1.
Table 1: List of primary antibodies used, and their incubation
details.
Antibody Species Clone ManufacturerIncubationtime (mins)
CD3 Rabbit 2GV6 Roche 16
CD8 Mouse C8/144b Dako 32
CD20 Mouse L26 Roche 16
PD1 Mouse NAT105 Abcam 60
PD-L1 Rabbit SP263 Roche 16
FOXP3 Mouse 236A/E7 Abcam 60
Following incubation with the first primary antibody, slides
wereincubated with a horseradish peroxidase labelled
secondaryantibody, followed by disclosure using
tyramide-linkedfluorescent marker. For duplex and multiplex
staining this wasfollowed by heat-induced epitope retrieval (HIER)
prior toaddition of the second primary antibody, followed by
incubationwith a horseradish peroxidase labelled secondary antibody
andfinally disclosure using the next tyramide-linked
fluorescentmarker. Fluorescent detection was performed using Opal
7-plexfluorophores (PerkinElmer, Waltham, Massachusetts, USA).Opal
fluorophores were supplied premixed with dimethylsulfoxide (DMSO)
and were diluted 1:75 with tyramide signalamplification fluid
(TSA). Details of the fluorophores used andthe primary antibodies
they were designated to for the 6-plexstain, are shown in Table 2
(note, different fluorophore-antibodymatches were used in the
single-plex and duplex stains). For the6-plex stain, following
secondary antibody and fluorescentmarker detection of the sixth
antibody, sections were mountedwith ProLong Gold Antifade Mountant
with DAPI (MolecularMolProbes, Eugene, Oregon, US) and cover
slipped. For thesingle-plex and duplex stains, DAPI (PerkinElmer)
was appliedmanually before mounting with ProLong Diamond
AntifadeMountant (ThermoFisher, Waltham, Massachusetts, US) and
cover slip addition.
2
listed in Table 1 and then chromogenic detection following
themanufacturer’s recommendation for each kit. Counterstaining
Syed J, et al.
Immunotherapy (Los Angel), Vol.5 Iss.2 No:1000157
-
Fluorophore
Excitationwavelength(nm)
Emissionwavelength(nm)
Designatedantigen
Opal520 494 525 CD3
Opal540 523 536 CD8
Opal570 550 570 FOXP3
Opal620 588 616 CD20
Opal650 627 650 PD1
Opal690 676 694 PD-L1
HALO image analysis of single-plex
chromogenicimmunohistochemistry to determine HIER condition
The single-plex slides for each antibody at each HIER
conditionwere digitalised using an Aperio AT2 whole slide scanner
(Leica)at 20x objective. The HALO® image analysis platform was
usedto quantitatively analyse the protein expression, with
stainsclassified as either cyto-nuclear, membrane or
immune.Consecutive tissue sections were co-registered using a
rigid-bodyregistration method within the HALO platform and
proteinexpression measured using colour deconvolution to
separateDAB and Hematoxylin staining based on optical density.
Thenumber of cells positive for each antibody was comparedbetween
different durations of HIER with linear regressionanalysis using
Graphpad Prism version 6.0 (Graphpad SoftwareInc, San Diego,
California, USA).
HALO Image analysis of single-plex and
dupleximmunofluorescence
Single-plex and duplex IF stained slides were digitalised
usingthe AxioScan Z1 (Zeiss). HALO® image analysis platform
incombination with the cytonuclear fluorescence algorithm wasused
to analyse protein expression of each fluorescent stainacross
serial tissue sections. The number of cells positive foreach
antibody was compared between different durations ofHIER with
linear regression analysis using Graphpad Prismversion 6.0.
Inform image analysis of multiplex immunofluorescence
Multispectral image analysis was performed using Informsoftware
version 2.1.1 (PerkinElmer, Waltham, Massachusetts,US). Cell-based
segmentation was used with nuclearsegmentation, based on DAPI
counterstaining. For Cohort 1(whole sections), five regions of
interest (ROI) were selectedfrom each DAB stained section and were
matched across single-plex fluorescent and multiplex stained
sections (Figure 2b). ForCohort 2 the entire TMA was imaged at x200
magnificationusing a Vectra multispectral microscope and after
cell
for the same antibody applied to the same TMA in single-plexIF.
A spectral library composed of the spectra of each of the sixOpal
fluorophores singly was generated, which could then beused together
with spectra of DAPI and tissue auto fluorescence.This was applied
with spectral un-mixing to generate positivity,followed by
phenotype training to identify cells positive for eachantibody. The
trained Inform protocol was applied to the fiveROI and the number
of positive cells for each antibody wascalculated between the
single-plex and multiplex stainedsections.
Statistical analysis
The strength of association between the numbers of cellspositive
for each antibody as indicated by single-plex andmultiplex sections
was assessed by linear regression (only for 6-plex experiment)
analysis using Graphpad Prism version 6.0. R2
values from regression analysis was used for the final 5
plexcomparisons. Coefficient of variability was determined
usingGraphpad Software for duplex IF and single-plex serial
sectionIF experiments.
RESULTS
Investigation of effect of antibody order in
multiplexfluorescent immunofluorescence
Prior to considering validation of a full multiplex IF protocol;
itwas first essential to demonstrate concordance between
single-plex chromogenic (DAB) IHC and single-plex IF
staining.Quantification of CD3, CD8 and FOXP3 on control
tonsiltissue demonstrated concordance between the two
stainingprotocols, which fell within expected intra-run
variability(Figures 1A-1F). Subsequently, the effect of the
position in amultiplex IF order for any given antibody was
investigated acrossa panel of six antibodies, selected to detect a
range of nuclearand membrane markers characteristic of infiltrating
immunecells. Specifically, these were CD3+ lymphocytes, CD20+ B
cells,CD8+ cytotoxic T cells, Foxp3+ regulatory T cells (Tregs),
PD1(program death 1) and PD-L1 (program death-ligand 1).
For each position in the multiplex staining order (1 to 6),
acomparison was made on serial sections, between the
changingmultiplex position and single-plex IF (Table 3). Five
regions withvariable immune cell densities were selected from whole
tissuesections of cohort 1, aligned across the slides being
compared.The single-plex methodologies were applied in this
multiplexsystem without further protocol optimisation.
Unfortunately,this resulted in PD-L1 run fails and this antibody
was thereforeomitted from analysis, leaving data for five of the
sixexperimental runs. For CD3, CD8, CD20, FOXP3 and PD1 theR2
values from regression analysis presented a trend toprogressive
reduction as the respective antibody advanced inposition (Figures
2A-2E). This indicates a reduction in fidelity ofantibody staining
as the antibody was applied progressively laterin the multiplex
staining order.
3
phenotyping and spectral un-mixing, the total number ofpositive
cells for each antibody were counted for each core anddivided by
the area of tissue in the core. The mean cell countsper tissue area
were then compared for each antibody with those
Table 2: Details of the fluorophores used and the primary
antibodiesthey were designated to for the 6-plex stain.
Immunotherapy (Los Angel), Vol.5 Iss.2 No:1000157
Syed J, et al.
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R2 figures for multiplex IF vs. Single-plex IF at the
followingpositions
Antibody Position 2 Position 3 Position 4 Position 5 Position
6
CD3 0.807 0.7 0.683 0.267 0.136 0.241
CD8 0.74 0.651 0.596 0.301 0.002 0.313
CD20 0.679 0.697 0.789 0.431 0.242 0.14
FOXP3 0.021 0.249 0.203 0.016 0.002 0.012
PD1 0.98 0.212 0.309 0.196 0.115 0.001
PD-L1 NA NA NA NA NA NA
Figure 1: Analysis and quantification of immune cell
biomarkerchromogenic (DAB) and immunofluorescence (IF) single-plex
stainsusing HALO image analysis: A, C and E) Representative images
andHALO image analysis mark-ups of CD3/CD8/FOXP3 respectively,using
DAB and IF staining on human tonsil tissue. B, D and F)HALO
quantification of CD3/CD8/FOXP3 positive cells/mm2 oftonsil tissue
respectively detected using DAB and IF staining.
Investigation of biological/run variability in serialsectioning,
antibody steric hindrance and HIERconditions in duplex IF
Several experimental hypotheses were tested to help
understandthe reasons behind the variability in antibody staining
observedin Figure 2. Through combination of membrane markers
(CD3
4
Table 3: Comparison on serial sections, between the
changingmultiplex position and single-plex IF.
Immunotherapy (Los Angel), Vol.5 Iss.2 No:1000157
Syed J, et al.
(IF) single-plex and multiplex staining: Scatter plots for
comparisonof positivity between a single-plex stained section and
positivity forthe same antibody in a multiplex stained section with
advancingorder of antibody in the multiplex protocol from first to
sixth.Scatter plots shown for A) CD3, B) CD8, C) CD20, D) PD1 and
E)FOXP3. R2 values from regression analysis are included.
Position 1
Figure 2: Comparison of positivity between
immunofluorescence
-
demonstrates the importance of staining order in a
multiplexassay, where in this case, CD8 must be stained in position
1 toreplicate single-plex quantification.
Added to this, application of antibodies that target the
samecells and sub-cellular compartment are thought to cause
sterichindrance due to the proximity of the antibody binding,
apotential problem for multiplexing membrane markers such asCD3 and
CD8 which have biological overlap of the antigen.Staining and
quantification of CD3 when duplexed with CD8(Figures 3G and 3H)
showed minimal variation at eitherposition 1 or 2 compared with
single-plex CD3 DAB or IF (CV6.99%). In contrast, when quantifying
CD8 in duplex with CD3(Figures 3I and 3J), a 90% drop in CD8
positive cells wasdetected when CD8 was placed at position 2 after
CD3. Thisresulted in a high CV across the experimental groups of
59.51%,which was not observed when CD8 was combined with
nuclearFOXP3 (Figure 3E), and suggests steric hindrance is a
problemfor CD8 when combined with CD3.
An additional consideration was the effect of the increasedHIER
needed to strip previously stained antibodies. Serialsections were
stained for each antibody as a single-plex DABassay, subjected to
HIER durations corresponding to theexposure times at positions 1
through to 6 (increments of 8minutes per position) and cells
quantified using image analysis(Figure 4). The effect of HIER
duration was non-linear and non-uniform across the range of
antibodies, with all the antibodiesshowing both a rise and fall in
immune cell counts as durationof HIER increased. Of note, the DAB
clinical protocol forCD20 doesn’t require HIER and subsequently
when HIER wasincluded, the staining specificity was completely lost
(Figure 4C).Due to this, CD20 was removed from this multiplex
protocoland classed as incompatible to multiplex. The HIER data
aresummarized in Figure 5.
A subsequent assay was developed to test the temperaturerequired
to achieve successful stripping, and if this could bereduced to
limit antibody impact. The cut-off for successfulremoval of
antibody conjugates was determined to be 72°C (datanot shown).
However, analysis of serial sections stained for CD8as single-plex
DAB, single-plex IF, or single-plex IF with anadditional round of
stripping revealed no statistical significancein CD8 detection
using 72°C rather than routinely used 95°C(Figure 5C). Reducing
stripping temperature therefore did not
Figure 3: HALO image analysis was used to assess the impact
ofstaining order and steric hindrance in immunofluorescence
(IF)duplex stains: A) Representative image and HALO image
analysismark-up of CD3/FOXP3 IF duplex staining on human tonsil
tissue,where CD3 (red) was stained in position 1 and FOXP3 (green)
wasin position 2. B) HALO quantification of CD3 positive
cells/mm2
tissue in chromogenic (DAB) CD3 single-plex, IF CD3
single-plexand IF duplex stains where CD3 was placed in either
position 1 or 2in combination with FOXP3. Percentage coefficient of
variation(CV) was calculated across the samples. C) Intra-run
variability ofCD3 IF stained serial tonsil sections; CD3 positive
cells/mm2 tissueand the CV are shown. D) Representative image and
HALO imageanalysis mark-up of CD8/FOXP3 IF duplex staining on
humantonsil tissue, where CD8 (orange) was stained in position 1
andFOXP3 (green) was in position 2. E) HALO quantification of
CD8positive cells/mm2 in DAB CD8 single-plex, IF CD8 single-plex
andIF duplex stains where CD8 was placed in either position 1 or 2
incombination with FOXP3. CV also shown. F) Intra-run variability
ofthe CD8 single-plex IF staining of three serial tonsil sections
stainedfor CD8 using IF (CV also shown). G) Representative image
andHALO image analysis mark-up of CD3/CD8 IF duplex staining
onhuman tonsil tissue, where CD3 (orange) was stained in position
1and CD8 (green) was in position 2. H) HALO quantification ofCD3
positive cells/mm2 tissue in DAB CD3 single-plex, IF CD3single-plex
and IF duplex stains where CD3 was placed in eitherposition 1 or 2
in combination with CD8. I) Representative imageand HALO image
analysis mark-up of CD8/CD3 IF duplex stainingon human tonsil
tissue, where CD8 (green) was stained in position 1and CD3 (orange)
was in position 2. J) HALO quantification ofCD8 positive cells/mm2
tissue in DAB CD3 single-plex, IF CD3single-plex and IF duplex
stains where CD8 was placed in eitherposition 1 or 2 in combination
with CD3.
5Immunotherapy (Los Angel), Vol.5 Iss.2 No:1000157
Syed J, et al.
sections stained with respective single-plex DAB, single-plex
IFand duplex IF (membrane marker at position 1 or 2) wascompared
with single-plex IF variability across three serialsections. The
coefficient of variability (CV) for single-plex IFserial sections
was comparable to duplex assay CV for CD3(Figures 3A-3C) but not
CD8 (Figures 3D-3F). Of note,detection of CD8 when placed in
position 2 saw a 1.6 foldincrease from single-plex IF
quantification. This again
rescue the over-retrieval effect of staining position on CD8,
withhigher CD8 levels quantified in the multi-plex compared to
thegold standard single-plex assays.
or CD8) with nuclear FOXP3 in duplex staining on controltonsil
tissue, we confirmed variability from staining order wasnot simply
attributable to biological heterogeneity between serialsections.
For both CD3 and CD8 assays, variability of four serial
-
Figure 4: The effect of Heat Induced Epitope Retrieval
(HIER)conditions on antibody staining was investigated using Inform
andHALO image analysis: Single-plex chromogenic (DAB) assays
wererun for A) CD3, B) CD8, C) CD20, D) PD1, E) FOXP3 and F) PD-L1
on tonsil tissue. The impact of increasing HIER time on
positivecell count was determined using serial sections stained for
eachbiomarker. Quantification was performed using Inform
imageanalysis software. Image analysis representative images are
shown(left panels). Graphs depicting change in immune cell number
foreach antibody with increase in HIER time are demonstrated
(centrepanels) with associated values (right panels).
Figure 5: Summary of the impact of Heat Induced Epitope
Retrieval(HIER) conditions on antibody staining and attempts to
resolve: A)Summary of the impact of increasing antigen retrieval
(AR) durationacross single-plex chromogenic (DAB) assays for CD3,
CD8, CD20,PD1, FOXP3 and PDL1 on tonsil tissue. B) Representative
imagesand HALO image analysis mark-up of CD8 DAB
andimmunofluorescence (IF) staining on human tonsil tissue. Two
IFstained slides were subjected to an additional HIER step at
either72°C or 95°C following incubation with the tyramide
linkedfluorophore. C) Comparison of CD8 positive cells/mm2 tissue
withDAB, IF single-plex and IF single-plex with additional HIER at
72°Cor 95°C. Quantification was performed using HALO image
analysiswith results presented as mean ± SEM from three
independentexperiments. CV calculated across the means of each
group.
Optimised multiplex IF protocol
Table 4: Single-plex DAB compared to single-plex IF resulted in
R2
correlations.
Antibodyfinal order
R2 figures for DABvs. Single-plex IF
R2 Figures for Single-plex IF vs.Multiplex IF
CD8 0.724 0.728
PD-L1 0.189 0.21
6
CD3 0.605 0.608
FOXP3 0.676 0.803
PD1 0.677 0.81
Immunotherapy (Los Angel), Vol.5 Iss.2 No:1000157
Syed J, et al.
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Figure 6: Comparison of biomarker positivity via
chromogenic(DAB), immunofluorescence (IF) single-plex and multiplex
stainsusing the final antibody order: Scatter plots for each
antibodyshowing comparison of positivity between single-plex
chromogenic(DAB) and single-plex IF serial stained sections (left
panels) andpositivity for the same antibody between single-plex IF
and multiplexIF serial stained sections (right panels), using
optimised multiplexorder of A) CD8, B) PD-L1, C) CD3, D) FOXP3 and
E) PD1.
Figure 7: Representative images for quantification of biomarkers
viachromogenic (DAB), immunofluorescence (IF) single-plex
andmultiplex stains: Representative images for each of the
antibodiesstained by single-plex chromogenic (left panels),
single-pleximmunofluorescence (middle panels) and
multipleximmunofluorescence (right panels), with, for
immunofluorescenceimages, false colour bright field images shown
below correspondingimmunofluorescence image. Images shown for a)
CD8, b) PD-L1, c)CD3, d) FOXP3 and e) PD1.
DISCUSSION
Multiplex IF staining is increasingly being used for
simultaneousanalysis of multiple biomarkers [9,14]. It is
particularly useful forinvestigation of TILS, which by their nature
require co-localisation of multiple markers for their
identification, makingmultiplex IF essential for their detection
[15-17]. Previous studieshave evaluated whether the staining for
any given antibody in amultiplex protocol equates qualitatively to
that when used singly
7
Building on the aforementioned data, a multiplex order
wassuggested of CD8, PD-L1, CD3, FOXP3 and PD1. Themultiplex IF
protocol detailed above was repeated with theantibodies applied in
this order using a tissue microarraycomposed of 100 cores of
lymphoid tissue containing follicularlymphoma (Cohort 2).
Single-plex DAB compared to single-plexIF resulted in R2
correlations ranging from 0.19 to 0.72 (Figure6 and Table 4). When
comparing single-plex IF to multiplex IFthe R2 correlations ranged
from 0.21 to 0.81, demonstrating agood correlation using the
optimised protocol. Representativeimages of the optimised protocol
staining are illustrated inFigure 7, showing single DAB,
single-plex IF, and multiplex IFand an associated IF artificial
bright field image for each marker.
Immunotherapy (Los Angel), Vol.5 Iss.2 No:1000157
Syed J, et al.
-
[18], but only recently have studies advanced into
understandingwhether multiplex staining recapitulates single-plex
stainingquantitatively or the impacting of antibody stripping
[19-22]. Inthe present study we quantitatively test the effect of
antibodyorder in multiplex staining, showing that it is not
feasible tosimply merge clinically validated single-plex DAB
protocols intoa multiplex methodology. As such, thorough
re-optimisation andvalidation will be required to ensure the
accuracy and sensitivityof any multiplex assays.
This study indicates, in methods where multiplexing is
enabledusing antigen retrieval to strip prior antibodies, that
theprincipal compounding factor influencing multiplex validationis
the effect of extended times of HIER as antibodies advance inorder
in a multiplex protocol. Sequential HIER steps occurbetween each
antibody and duration of HIER had a qualitativeeffect on immune
cell counts for all antibodies, the effect beingnon-linear and
non-uniform, with all the antibodies showingboth rise and fall in
immune cell counts as duration of HIERincreased. A reduction of the
HIER temperature did not resolvethe impact on susceptible
biomarkers such as CD8. These dataenabled design of an optimised
antibody order for the multiplexIF protocol, with minimisation of
the effect of increasingduration of HIER by placing antibodies more
affected earlier inthe multiplex order.
The final optimised antibody order was CD8, PD-L1, CD3,FOXP3,
and PD1. These results demonstrated valid substitutionof
chromogenic staining for single-plex IF and that
sequentialmultiplexing on a single tissue section can be
representative ofthe single-plex staining for several antibodies,
but only if theeffect of order in the multiplex protocol is
considered. Theorder should be tested for each antibody combination
used, toenable high concordance between single-plex and
multiplexdata.
CONCLUSION
In conclusion, an optimised multiplex IF protocol was devised,in
which correlation between multiplex antibody staining
andsingle-plex staining was maximised. Knowledge that
multiplexprotocols can reach concordance with clinically validated
single-plex chromogenic assays is of great importance.
Clinicalpathology presently relies on single-plex chromogenic
staining,whilst multiplex IF will be important as personalised
medicineincreasingly entails assessment of multiple
biomarkers,preferably simultaneously. Multiplex IF will conserve
limitedclinical material and enable spatial resolution of
co-localisedantigens to facilitate numeration of complex immune
cell sub-populations, which require several markers for
theiridentification. These data will increase our understanding of
themolecular mechanisms involved in the anti-tumour
immuneresponse.
CONFLICTS OF INTEREST
HKA, GNJ, AL, AS are employees and shareholders ofAstraZeneca.
Collaborative Research Agreement with Perkin-Elmer and Definiens
(of relevance to this manuscript). WH isan employee and shareholder
of Abcam PLC and former
employee of AstraZeneca. JS, JA, JJ, CS, and RB have
noconflicts.
REFERENCES
1. Angell H, Galon J. From the immune contexture to
theImmunoscore: The role of prognostic and predictive immunemarkers
in cancer. Curr Opin Immunol. 2013;25: 261-267.
2. Galon J, Fridman WH, Pages F. The adaptive
immunologicmicroenvironment in colorectal cancer: A novel
perspective.Cancer Res. 2007;67: 1883-1886.
3. Fridman WH, Pages F, Sautes-Fridman C, Galon J. The
immunecontexture in human tumours: Impact on clinical outcome.
NatRev Cancer. 2012;12: 298-306.
4. Gooden MJ, de Bock GH, Leffers N, Daemen T, Nijman HW.The
prognostic influence of tumour-infiltrating lymphocytes incancer: A
systematic review with meta-analysis. Br J
Cancer.2011;105(1):93-103.
5. Anitei MG, Zeitoun G, Mlecnik B, Marliot F, Haicheur N,
TodosiAM, et al. Prognostic and predictive values of the
immunoscore inpatients with rectal cancer. Clin Cancer Res.
2014;20: 1891-1899.
6. Galon J, Pages F, Marincola FM, Angell HK, Thurin M, Lugli
A,et al. Cancer classification using the Immunoscore: A
worldwidetask force. J Transl Med. 2012;10: 205.
7. Bobrow MN, Shaughnessy KJ, Litt GJ. Catalyzed
reporterdeposition, a novel method of signal amplification. II.
Applicationto membrane immunoassays. J Immunol Methods.
1991;137:103-112.
8. Levenson RM, Mansfield JR. Multispectral imaging in biology
andmedicine: Slices of life. Cytometry Part A. Cytometry
A.2006;69:748-758.
9. Stack EC, Wang C, Roman KA, Hoyt CC.
Multiplexedimmunohistochemistry, imaging, and quantitation: A
review, withan assessment of Tyramide signal amplification,
multispectralimaging and multiplex analysis. Methods. 2014;70:
46-58.
10. Van der Loos CM. Multiple immunoenzyme staining: Methodsand
visualizations for the observation with spectral imaging.
JHistochem Cytochem. 2008;56: 313-328.
11. Ward MJ, Thirdborough SM, Mellows T, Riley C, Harris
S,Suchak K, et al. Tumour-infiltrating lymphocytes predict
foroutcome in HPV-positive oropharyngeal cancer. Br J
Cancer.2014;110: 489-500.
12. Huang W, Hennrick K, Drew S. A colorful future of
quantitativepathology: Validation of Vectra technology using
chromogenicmultiplexed immunohistochemistry and prostate
tissuemicroarrays. Human Pathology. 2013;44: 29-38.
13. Halse H, Colebatch AJ, Petrone P, Henderson MA, Mills JK,
SnowH, et al. Multiplex immunohistochemistry accurately defines
theimmune context of metastatic melanoma. Scientific
Reports.2018;8: 11158.
14. Dixon AR, Bathany C, Tsuei M, White J, Barald KF, Takayama
S.Recent developments in multiplexing techniques
forimmunohistochemistry. Expert Rev Mol
Diagn.2015;15:1171-1186.
15. Feng Z, Jensen SM, Messenheimer DJ, Farhad M, Neuberger
M,Bifulco CB, et al. Multispectral imaging of T and B cells in
murinespleen and tumor. J Immunol. 2016;196: 3943-3950.
16. Nowicki TS, Akiyama R, Huang RR, Shintaku IP, Wang X,Tumeh
PC, et al. Infiltration of CD8 T Cells and Expression ofPD-1 and
PD-L1 in Synovial Sarcoma. Cancer Immunol Res.2017;5: 118-126.
17. Schalper KA, Carvajal-Hausdorf D, McLaughlin J, Altan
M,Velcheti V, Gaule P, et al. Differential expression and
significance
8Immunotherapy (Los Angel), Vol.5 Iss.2 No:1000157
Syed J, et al.
-
of PD-L1, IDO-1, and B7-H4 in human lung cancer. Clin CancerRes.
2017;23: 370-378.
18. Carvajal-Hausdorf DE, Schalper KA, Neumeister VM, Rimm
DL.Quantitative measurement of cancer tissue biomarkers in the
laband in the clinic. Lab Invest. 2015;95: 385-396.
19. Parra ER, Uraoka N, Jiang M, Cook P, Gibbons D, Forget MA,
etal. Validation of multiplex immunofluorescence panels
usingmultispectral microscopy for immune-profiling of
formalin-fixedand paraffin-embedded human tumor tissues. Sci Rep.
2017;7:13380.
20. Gorris MAJ, Halilovic A, Rabold K, van Duffelen
A,Wickramasinghe IN, Verweij D, et al. Eight-color multiplex
immunohistochemistry for simultaneous detection of
multipleimmune checkpoint molecules within the
tumormicroenvironment. J Immunol. 2018;200: 347-354.
21. Surace M, DaCosta K, Huntley A, Zhao W, Bagnall C, Brown
C,et al. Automated multiplex immunofluorescence panel
forimmuno-oncology studies on formalin-fixed carcinoma
tissuespecimens. J Vis Exp. 2019;28: 158-163.
22. Bolognesi MM, Manzoni M, Scalia CR, Zannella S, Bosisio
FM,Faretta M, et al. Multiplex staining by sequential
immunostainingand antibody removal on routine tissue sections. J
HistochemCytochem. 2017;65: 431-444.
9Immunotherapy (Los Angel), Vol.5 Iss.2 No:1000157
Syed J, et al.
内容Multiplex Immunohistochemistry: The Importance of Staining
Order When Producing a Validated
ProtocolABSTRACTINTRODUCTIONMATERIALS AND METHODSTissue cohorts and
ethicsChromogenic single-plex immunohistochemistry stainingStaining
of single-plex, duplex and multiplex immunofluorescenceHALO image
analysis of single-plex chromogenic immunohistochemistry to
determine HIER conditionHALO Image analysis of single-plex and
duplex immunofluorescenceInform image analysis of multiplex
immunofluorescenceStatistical analysis
RESULTSInvestigation of effect of antibody order in multiplex
fluorescent immunofluorescenceInvestigation of biological/run
variability in serial sectioning, antibody steric hindrance and
HIER conditions in duplex IFOptimised multiplex IF protocol
DISCUSSIONCONCLUSIONCONFLICTS OF INTERESTREFERENCES